Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

Summary

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

Abstract

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Introduction

Eukaryotic DNA is organized and compacted by histone proteins, forming chromatin. The nucleosome core particle (NCP) constitutes the fundamental repeating unit of chromatin that regulates accessibility to DNA-binding proteins for DNA repair, transcription, and replication¹. Although the first X-ray crystal structure of the NCP was first solved more than two decades ago² and many more structures of the NCP have been published since^3,4,5,6, DNA repair mechanisms in nucleosomal substrates have not....

Protocol

1. Assemble nucleosome core particles via salt-gardient dialysis

NOTE: The preparation of nucleosome core particles using recombinant histone proteins for structural studies has been extensively described in detail by others^14,15,16. Follow the purification of recombinant X. laevis histones and histone octamer assembly described by others^14,15, and assemble the nucleosomal substrate as described below.

1. Purchase three oligonucleotides (listed in ....

Results

Properly assembled NCPs (Figure 2) were used to make a complex with a recombinant fusion protein of MBP-Polβ-APE1 (Figure 3). To determine the ratio of NCP to MBP-Polβ-APE1 to form a stable complex, we performed electrophoretic mobility shift assays (EMSA) (Figure 4), which showed a singly shifted band of the NCP with 5-fold molar excess of MBP-Polβ-APE1. During the optimization of making this complex, crosslinking wi.......

Discussion

A specific protocol for purifying the DNA repair factor will be dependent on the enzyme of interest. However, there are some general recommendations, including the use of recombinant methods for protein expression and purification¹⁸; if the protein of interest is too small (<50 kDa), structure determination by cryo-EM had been nearly impossible until more recently through the use of fusion systems¹⁹, nanobody-binding scaffolds²⁰, and optimizing i.......

Materials

Name	Company	Catalog Number	Comments
1 M HEPES; pH 7.5	Thermo Fisher Scientific	15630080
1 M MgCl2	Thermo Fisher Scientific	AM9530G
10x TBE	Bio-rad	1610733
25% glutaraldehyde	Fisher Scientific	50-262-23
3 M KCl	Thermo Fisher Scientific	043398.K2
491 prep cell	Bio-rad	1702926
Amicon Ultra 15 centrifugal filter (MW cutoff 30 kDa)	Millipore Sigma	Z717185
Amicon Ultra 4 centrifugal filter (MW cutoff 30 kDa)	Millipore Sigma	UFC8030
AutoGrid Tweezers	Ted Pella	47000-600
Automatic Plunge Freezer	Leica	Leica EM GP
C-1000 touch thermocycler	Bio-rad	1851148
C-clips and rings	Thermo Fisher	6640--6640
Clipping station	SubAngostrom	SCT08
Dialysis Membrane (MW cufoff 6-8 kDa)	Fisher Scientific	15370752
Diamond Tweezers	Techni-Pro	758TW0010
dsDNA	Integrated DNA techonologies	N/A
FEI Titan Krios	Thermo Fisher	KRIOSG4TEM
FPLC purification system	AKTA Pure	29018224
Fraction collector Model 2110	Bio-rad	7318122
Glow Discharge Cleaning System	Ted Pella	91000S
Grid Boxes	SubAngostrom	PB-E
Grid Storage Accessory Pack	SubAngostrom	GSAX
Liquid Ethane	N/A	N/A
Liquid Nitrogen	N/A	N/A
Minipuls 3 peristaltic two-head pump	Gilson	F155008
Nanodrop	Thermo Fisher Scientific	ND-2000
Novex 16%, Tricine, 1.0 mm, Mini Protein Gels	Thermo Fisher Scientific	EC6695BOX
Pipetman	Gilson	FA10002M
Pipette tips (VWR) Low Retention	VWR	76322-528
Polyacrylamide gel solution (37.5:1)	Bio-rad	1610158
polyethylene glycol (PEG)	Millipore Sigma	P4338-500G
Pur-A-lyzer Maxi 3500	Millipore Sigma	PURX35050
Purified recombinant DNA repair factor	N/A	N/A
R 1.2/1.3 Cu 300 mesh Grids	Quantifoil	N1-C14nCu30-01
Recombinant histone octamer	N/A	N/A
Spring clipping tools	SubAngostrom	CSA-01
Superdex 200 column 10/300	Millipore Sigma	GE28-9909-44
Transmission Electron Microscope	Thermo Fisher	Talos Arctica 200 kV
Tweezers Assembly for FEI Vitrobot Mark IV-I	Ted Pella	47000-500
UltraPure Glycerol	Thermo Fisher Scientific	15514011
Vitrobot	Thermo Fisher	Mark IV System
Whatman Filter paper (55 mM)	Cytiva	1005-055
Xylene cyanol	Thermo Fisher Scientific	440700500
Zeba Micro Spin Desalting Columns, 7K MWCO, 75 µL	Thermo Fisher Scientific	89877